Preparation of low molecular weight polylysine and polyornithine in high yield

ABSTRACT

The present invention generally relates to the large-scale (e.g., greater than 1 kg scale) preparation of low molecular weight polylysine or polyornithine in high yield by preparing a polylysine or polyornithine having a weight average molecular weight from about 12,500 Daltons to about 22,000 Daltons and hydrolyzing it to produce a polylysine or polyornithine having a weight average molecular weight from about 5,500 Daltons to about 12,000 Daltons. In preferred embodiments, the polymer is a homopolymer of poly-L-lysine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the United States national stage of InternationalApplication No. PCT/US2009/052085, filed Jul. 29, 2009, which claims thebenefit of U.S. Provisional Application No. 61/087,031, filed Aug. 7,2008, the entire content of each of which is hereby incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention generally relates to the preparation oflarge-scale (greater than 1 kg scale) low molecular weight polylysine orpolyornithine in high yield by preparing a polylysine or polyornithineintermediate having a lysine- or ornithine-basis weight averagemolecular weight from about 12,500 Daltons to about 22,000 Daltons andhydrolyzing it to produce a polylysine or polyornithine having a weightaverage molecular weight from about 5,500 Daltons to about 12,000Daltons. In preferred embodiments, the process is used to preparepolylysine. In some of these embodiments, the polymer is a homopolymerof poly-L-lysine.

BACKGROUND OF THE INVENTION

Polylysine and polyornithine are known and are under investigation asDNA condensing agents to be used in non-viral gene therapy. However, asis known in the literature, poly-L-lysine has cytotoxic effects thatdecrease with decreasing molecular weight. Thus, preparation of a lowmolecular weight polylysine is desirable.

Poly-L-lysine and other polyamino acids have been prepared by a varietyof routes. Commercially, poly-L-lysine has been prepared by reaction ofN^(ε)-carbobenzyloxy(Cbz)-L-Lysine, N-carboxyanhydride with apolymerization initiator to produce poly-N^(ε)-Cbz-L-lysine. Thepoly-N^(ε)-Cbz-L-lysine is treated with hydrobromic acid in acetic acidto remove the Cbz group and form poly-L-lysine having a molecular weightof about 7,000 Daltons. The product can be dialyzed, lyophilized, andrecovered in an 11-30% yield. This yield of the current process isvariable and is unacceptably low. Thus, a need exists for higher yieldlarge-scale processes having less variability to produce polylysinehaving a lysine-basis weight average molecular weight from about 5,500Daltons to about 12,000 Daltons.

SUMMARY OF THE INVENTION

Among the various aspects of the invention is a process for preparing alarge-scale relatively low molecular weight polylysine or polyornithinein high yield.

Another aspect of the invention is a process for the preparation of apolylysine or polyornithine polymer comprising repeating unitscorresponding to Formula 3 or a salt thereof and having a lysine- orornithine-basis weight average molecular weight from about 5,500 toabout 12,000 Daltons. The process comprises hydrolyzing a polylysine orpolyornithine intermediate comprising repeating units corresponding toFormula 3 or a salt thereof and having a lysine- or ornithine-basisweight average molecular weight from about 12,500 to about 22,000,wherein Formula 3 has the structure:

and n is 3 or 4.

The process of the invention described above further comprises removingthe nitrogen protecting group from a protected polylysine orpolyornithine intermediate comprising repeating units of Formula 2 or asalt thereof and having a lysine- or ornithine-basis weight-averagemolecular weight of from about 12,500 Daltons to about 22,000 Daltons,wherein Formula 2 has the structure

R₁ is an amino protecting group; and n is 3 or 4.

The various processes described above can further comprise preparationof the protected polylysine or polyornithine intermediate bypolymerizing an N-carboxyanhydride compound of Formula 1, whereinFormula 1 has the structure

R₁ is an amino protecting group; and n is 3 or 4.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Advantageously, polylysine or polyornithine can be prepared inaccordance with the process of the present invention in a relativelymore efficient and/or productive manner. In general, the polylysine orpolyornithine polymer comprises repeating units corresponding to Formula3, N^(ε)-salts, or N⁶⁷-salts thereof:

wherein n is 3 (i.e., polyornithine) or 4 (i.e., polylysine). Thepolylysine or polyornithine polymer product comprises a mixture ofmolecules having repeating units of Formula 3 to form a polymer having alysine- or ornithine-basis weight average molecular weight of from about5,500 Daltons to about 12,000 Daltons.

Generally, the homopolymers of polylysine can be comprised of D-, L-, ora racemic mixture of isomers of the lysine repeat units. In preferredembodiments, the polylysine comprises poly-L-lysine. Further, thehomopolymers of polyornithine can be comprised of D-, L-, or a racemicmixture of isomers of the ornithine repeat units. In preferredembodiments, the polyornithine comprises poly-L-ornithine. Thestereochemistry of the polylysine or polyornithine is determined by thestereochemistry of the starting monomer. The structures of Formulae 1,2, and 3 represent monomers and polymers having a D-, L-, or a mixtureof D- and L-stereochemistry.

Regardless of the composition of the repeat units, the process of thepresent invention enables the preparation of polylysine having a weightaverage lysine-basis molecular weight of from about 5,500 Daltons toabout 12,000 Daltons. For certain applications, polylysine having anaverage lysine-basis molecular weight of about 7,000 Daltons to about10,000 Daltons are desired. The process also enables the preparation ofpolyornithine having a weight average ornithine-basis molecular weightof from about 5,500 Daltons to about 12,000 Daltons.

For purposes of this application, the weight average molecular weight isdetermined using the gel permeation chromatography with multi-anglelaser light scattering (GPC MALLS) technique that provides an absoluteweight average molecular weight and polydispersity index.

Polymerization

The polylysine or polyornithine of the present invention may be preparedby a process that begins with polymerization of a N-carboxyanhydride ofan N^(ε)-protected lysine or an N⁶⁷-protected ornithine, in apolymerization medium comprising a suitable solvent. Polymerization ispreferably conducted in the presence of a polymerization initiatorcontained in the reaction medium.

The preparation of N-carboxyanhydrides of protected lysines andornithines is known. In general, the protected lysine or ornithine istreated with phosgene in an ethereal solvent (e.g., tetrahydrofuran) toproduce the corresponding N^(ε)-protected-lysine, N-carboxyanhydride orN⁶⁷-protected-ornithine, N-carboxyanhydride. In certain embodiments, theN^(ε)-protected-lysine, N-carboxyanhydride or N⁶⁷-protected-ornithine,N-carboxyanhydride corresponds to Formula 1

wherein R₁ is amino protecting group and n is 3 or 4. R₁, for example,can be carbobenzyloxy (Cbz), t-butyloxycarbonyl (t-Boc), orallyloxycarbonyl. Preferably, R₁ is carbobenzyloxy (Cbz). Thus, forexample, in one embodiment, the N-carboxyanhydride is theN-carboxyanhydride of Cbz-lysine or Cbz-ornithine and corresponds toFormula 1A.

In general, the polymerization initiator is a nucleophile. In addition,the polymerization initiator preferably possesses physical properties,which enable the initiator to be separated from the product polymer orotherwise eliminated from the reaction mixture upon the completion ofthe polymerization reaction. Exemplary polymerization initiators includemetal alkoxides having the formula MOR, wherein M is a metal and R is analkyl group. For example, the metal may be sodium or potassium. By wayof further example, the alkyl moiety may be a linear, branched or cyclicgroup having 1 to 10 carbon atoms. Presently preferred polymerizationinitiators include sodium methoxide, sodium ethoxide, sodium propoxide,potassium methoxide, potassium ethoxide, potassium propoxide, orcombinations thereof. Sodium methoxide is presently particularlypreferred.

The molar ratio of the N-carboxyanhydride (“NCA”) to the polymerizationinitiator used to form the polymerization reaction mixture may vary overa relatively wide range. It is generally preferred that the molar ratioof the NCA to the initiator be in the range of about 5:1 to about 15:1;preferably, about 8:1 to about 12:1, respectively.

The solvent used in the polymerization reaction mixture may generally beany suitable solvent. Exemplary solvents include dioxane, chloroform,dichloromethane, acetonitrile, and combinations thereof. Preferably, theinitial N-carboxyanhydride monomer is mixed with the solvent to providea monomer concentration between about 0.2 M and about 1 M; preferablyabout 0.4 M to about 0.6 M; or between about 0.02 wt. % to about 0.13wt. %; preferably, between about 0.05 wt. % to about 0.1 wt. %.

The reaction temperature of the polymerization of the N-carboxyanhydrideis not narrowly critical. The reaction may be carried out over a rangeof temperatures and times depending on the solvent used. The reactionconditions (e.g., solvent, pressure, and temperature) are selected inorder to provide a reaction rate that is not too slow and to minimizeloss of solvent due to evaporation. For example, polymerization may becarried out for a period of about 12 to about 30 hours, more typicallyabout 18 hours to 24 hours, at a temperature of about 20° C. to about40° C., more typically about 25° C. to about 30° C., and preferably atabout 25° C.

The resulting polymer contains protected lysine or ornithine repeatingunits. For example, these repeat units may generally correspond toFormula 2

wherein R₁ and n are as defined above in connection with Formula 1. Invarious preferred embodiments, when the repeating unit is a protectedlysine, the amino protecting group is Cbz, the protected polylysinerepeating unit generally corresponds to Formula 2A.

Upon completion of polymerization, the polyamino acid can beprecipitated in water and filtered, but typically the polymerizedprotected polymer is diluted with solvent to a concentration of about0.05 M to about 0.5 M (or about 0.0064 wt. % to about 0.064 wt. %)protected polylysine intermediate (based on the amount of the NCAstarting material); preferably, from about 0.1 M to about 0.3 M. Theproduct of the polymerization is a protected polylysine or polyornithineintermediate, preferably a protected lysine homopolymer, typicallyhaving a lysine-basis weight average molecular weight between about12,500 and about 22,000 Daltons.

Removing the Amino Protecting Group

The amino protecting group can be removed by catalytic hydrogenation ortreatment with a strong acid. These strong acids can include hydrobromicacid, hydrochloric acid, hydroiodic acid, sulfuric acid, and the like.In various preferred embodiments, the amino protecting group is removedby treatment with anhydrous hydrobromic acid. When the amino protectinggroup is removed, the polylysine or polyornithine contains repeat unitsof Formula 3

wherein n is as defined above in connection with Formula 1. In general,the number of repeating units varies depending the specific processconditions. Under the conditions prevailing in the deprotectionreaction, the number of repeating units provides a lysine-basis orornithine-basis weight average molecular weight from about 12,500Daltons to about 22,000 Daltons.

In the removal of the amino protecting group, the N^(ε)-substitutedpolylysine or N⁶⁷-substituted polyornithine intermediate is preferablycontacted with a strong acid. Where the polymerization reaction has beenconducted in a solvent medium, this solution may be diluted to provide areaction medium for the deprotection step. The polymerization reactionproduct is preferably diluted with the same solvent that has been usedfor the polymerization, to produce a solution having a concentration ofabout 0.05 M to about 0.5 M; preferably from about 0.1 M to about 0.3 M(or about 0.0064 wt. % to about 0.064 wt. %) protected polylysineintermediate (based on the amount of the NCA starting material) for usein the deprotection step. Alternatively, the protected polylysine orpolyornithine intermediate may have been precipitated from thepolymerization reaction solution and redissolved in the desired solventto produce a solution in the latter concentration range preparatory tothe deprotection step.

To carry out the deprotection reaction, the strong acid in a solvent oralone, may be introduced into the deprotection reaction medium.Preferably, the strong acid is introduced into the deprotection reactionmedium in a weight ratio to the Nε-protected polylysine or N⁶⁷-protectedpolyornithine (based on the NCA starting material) between about 2:1 andabout 4:1. In some cases, a solution of a strong acid comprising betweenabout 20 wt. % and about 40 wt. % strong acid (e.g., HBr) and betweenabout 60 wt. % and about 80 wt. % solvent (e.g., acetic acid) andintroduced into the polymerization medium. Preferably, a 30 to 35 wt. %hydrobromic acid in acetic acid solution is used as the source of HBr;this mixture typically contains between 0.5 and 1.5 wt. % water.Typically, the strong acid is preferably added relatively slowly, e.g.,over a period of about 45 to about 90 minutes (essentially dropwise on alaboratory scale), and the polymerization reaction mixture is agitatedto assure uniform mixing.

The reaction temperature of the deprotection step is not narrowlycritical. Preferably, the reaction is conducted at a temperature about20° C. to about 40° C.; more preferably, at about 25° C. to about 30°C.; typically at about 25° C. Typically, the deprotection step requiresapproximately 15 to 20 hours, most typically 18 hours of reaction.

The deprotection step produces the polylysine or polyornithineintermediate in the form of an N^(ε)- or N⁶⁷-mineral acid salt, whichprecipitates from the mixed organic deprotection reaction medium. Thissalt is separated from the supernatant liquid, as by filtration, and thesolids washed with an organic solvent, preferably a polar solvent suchas acetone, to remove the major side product of benzyl bromide. Thedeprotected polylysine or polyornithine intermediate salt or its freebase may then be taken up in an aqueous medium and hydrolyzed to yieldthe polylysine or polyornithine polymer product. Where a strong acid isused for the hydrolysis, the polylysine polymer product is also producedin the form of its N^(ε) or N^(δ) mineral acid salt.

Even after the solvent wash, the deprotection reaction product typicallyremains contaminated with oligomers, lower molecular weight salts andother low molecular weight by-products. Prior to the hydrolysisreaction, the deprotection reaction product may be further purified bytaking it up into an aqueous medium and subjecting it to dialysis orultrafiltration. A dialysis feed mixture comprising an aqueous solutionof the deprotection reaction product, e.g., in a concentration betweenabout 0.5 and about 1.5 wt. %, is placed in contact with a surface ofdialysis membrane, or an ultrafiltration membrane that has a carrierliquid, e.g., a flowing deionized water stream, in contact with itsopposite face. For ultrafiltration, a pressure differential of betweenabout 20 psi (138 kPa) and about 30 psi (207 kPa) may be establishedacross the membrane to promote flow of a permeate comprising lowmolecular weight components and solvent through the membrane to thecarrier solution. Transport of oligomers and low molecular weightcontaminants to the carrier liquid leaves a retentate comprisingdeprotected polylysine intermediate of reduced oligomer content.

The retentate may be diluted with water, preferably deionized, toprovide a solution typically comprising 0.03 wt. % to a 0.1 wt. %deprotected polylysine that may be used as the feed material to thehydrolysis step. Alternatively, the retentate may be lyophilized toyield a dry solid deprotected polylysine, which may then be redissolvedin an aqueous medium to provide conveniently a solution of the aforesaidconcentration that is sent to the hydrolysis step.

Hydrolysis to Reduce the Polymer Molecular Weight

Hydrolysis is conducted by contacting the deprotected polylysineintermediate in an aqueous hydrolysis reaction medium with a strong acid(e.g., hydrochloric acid, hydrobromic acid; hydroiodic acid, sulfuricacid, and the like) preferably having a concentration between about 0.1M and about 0.5 M in the aqueous medium. The ratio of strong acid todeprotected polylysine intermediate for the hydrolysis is preferablybetween about 1:5 and about 1:1. To establish the desired strong acidconcentration in the reaction medium, a strong acid source such as HBrmay conveniently be introduced into the hydrolysis reaction medium in asource solution having a concentration in the range of 40 wt. % to 68wt. %. Hydrolysis is preferably conducted at a temperature between about25° C. and about 30° C. and produces a polylysine or polyornithinepolymer having a lysine- or ornithine-basis weight average molecularweight from about 5,500 Daltons to about 12,000 Daltons from polylysineor polyornithine having a lysine- or ornithine-basis weight averagemolecular weight from about 12,500 Daltons to about 22,000 Daltons.Because the hydrolysis reaction proceeds progressively as long as thepolylysine or polyornithine remains in contact with the strong acidunder the aforesaid conditions, there is a need to establish the endpoint of the reaction so that the lysine- or ornithine-basis weightaverage molecular weight of the polylysine or polyornithine polymerproduct falls within the target range, preferably 5,500 to 12,000Daltons. In accordance with the process of the invention, the end pointof the hydrolysis reaction may be estimated based on the viscosity of asample solution of dialyzed and lyophilized deprotected polylysine orpolyornithine intermediate. If the entire quantity of deprotectedpolylysine or polyornithine intermediate has been purged of oligomersand low molecular weight contaminants by dialysis and the retentate thenlyophilized, a sample of this lyophilized intermediate can be used forthe end point projection. Otherwise, a sample of the unpurifieddeprotected polylysine or polyornithine intermediate may be taken andsubjected to dialysis and lyophilization on a laboratory scale toprovide the specimen used in end point projection.

To estimate the desired hydrolysis reaction time, a test solution isprepared consisting of a 1% by lysine- or ornithine-basis weight aqueoussolution of the dialyzed and lyophilized sample or specimen of dialyzedand lyophilized deprotected polylysine or polyornithine intermediate,and the viscosity of this solution is determined at 25° C. The viscosityof the sample is determined by adding 10 mL of the solvent to aCannon-Fenske, size 50 viscometer, placing the viscometer in a constanttemperature water bath having a temperature of 25±0.1° C. and verticallyaligning the capillary tube of the viscometer with a weighted thread onthe outside of the bath. The viscometer temperature is allowed to reachequilibrium over 20 minutes, the solvent is drawn into the tube to justabove mark M (see FIG. 1). The time in seconds for the meniscus to passfrom mark M to mark N is the efflux time, t₀, for the solvent. At leastfour successive readings are made of the flow time until the averagedeviation from the mean is less than ±0.1 second. The viscometer iscleaned and the process above is repeated using the 1 wt. % aqueoussolution of the polymer product to get the efflux time, t, for thesample. The relative and specific viscosities are calculated using thefollowing equations.Relative viscosity(η_(r))=t/t ₀Specific viscosity(η_(sp))=η_(r)−1

To calculate the viscosity molecular weight, the equation below is used(see A. Aaron and A. Berger, Biochim. Biophys. Acta, volume 69, page 397(1963)). This equation was developed for polylysine, but polylysine andpolyornithine are chemically similar with polyornithine having one fewermethylene group in its repeat unit, the same equation can be used.Degree of Polymerization(DP)=log⁻¹[log(η_(sp) /c)×0.79+2.46]The variable, η_(sp), is the specific viscosity and c is theconcentration of the polymer solution. The molecular weight iscalculated using the following equations.Molecular weight=DP×209(for Polylysine.HBr)Molecular weight=DP×195(for Polyornithine.HBr)

The time required to hydrolyze the polymer to the desired weight averagemolecular weight of 5,500 to 12,000 Daltons (measured by GPC MALLS) maythen be calculated using the following formula:Time required=[(Viscosity Molecular Weight of unhydrolyzedpolymer−21,000)×0.012]+72(±10%) Hours.The 21,000 and 0.012 are factors that have been determined empirically.It will be understood that selection of 1 wt. % as the concentration ofthe polylysine or polyornithine in the test solution is arbitrary andthat any concentration that yields a reasonable viscosity versusmolecular weight gradient is acceptable. However, the constants in thereaction time algorithm set forth above apply only to the case of a 1wt. % solution. For any other concentration the constants of theequation would need to be determined by correlation of empirical data.

Once the time of hydrolysis is determined, the unhydrolyzed polymer isdissolved in deionized water to form a clear solution, mixed for 10minutes, and the strong acid is added and mixed for the calculated time.Typically, the reaction time for the hydrolysis reaction is from about100 hours to about 350 hours. The hydrolysis reaction is carried out tothe extent required to produce a polymer having a repeat unit of Formula3 and a weight average molecular weight of from about 5,500 Daltons toabout 12,000 Daltons. The polydispersity index (PDI) is calculated bydividing the weight average molecular weight by the number averagemolecular weight. The PDI indicates the distribution of individualmolecular masses in a batch of polymers. The PDI for polylysine made bythe process described above is 1.2 to 1.5.

After a polylysine or polyornithine product having the desired lysine-or ornithine-basis weight average molecular weight has been formed, thisproduct may be further purified by dialysis or ultrafiltration. Toprepare for dialysis, the hydrolysis reaction solution may be dilutedwith water in a volumetric ratio between 30 parts hydrolysis reactionsolution and 70 parts water to 70 parts hydrolysis reaction solution and30 parts water. Preferably the ratio is between 40:60 and 60:40,conveniently about 50:50. The dilute solution is then placed in contactwith one surface of a dialysis or ultrafiltration membrane while anaqueous carrier solution is placed in contact with the opposite side ofthe membrane. For ultrafiltration, a pressure differential between about20 psi (138 kPa) and about 30 psi (207 kPa) may be established acrossthe membrane to promote flow of a permeate comprising low molecularweight components and solvent through the membrane to the carriersolution. The dialyzed product is collected and preferably lyophilizedto yield the polylysine or polyornithine having repeat units of Formula3 in its commercially acceptable form.

The yield of the hydrolyzed polylysine having a lysine-basis weightaverage molecular weight from about 5,500 to about 12,000 Daltons istypically greater than about 45% based on the amount of the NCA startingmaterial. In certain embodiments, the yield of the hydrolyzed polylysinehas been found to range from about 45% to about 55% based on the amountof the NCA starting material. It has been observed that experiments toproduce polylysine having a lysine-basis weight average molecular weightfrom about 5,500 to about 12,000 Daltons in the polymerization steprequired more polymerization initiator and provided a lower yield.Without being bound by theory, it is hypothesized that the greaterconcentration of polymerization initiator initiated growth of morepolymer chains than with a lower concentration of polymerizationinitiator and consequently, because the NCA monomer could add torelatively more growing chains, the polymer product had many chains thatwere of a lysine-basis weight average molecular weight below the desired5,500 Daltons. The necessary removal of these, as by dialysis orultrafiltration, resulted in poor yields. By comparison, when theprocess of the present invention is used to prepare a higher molecularweight polymer that is hydrolyzed to provide the desired weight averagemolecular weight, a lower concentration of polymerization initiator isused in the polymerization of the NCA and there is a smaller amount ofundesirably low molecular weight polymer products formed. Further, ithas been observed that longer polymer chains are hydrolyzed first andthat control of the hydrolysis reaction time can empirically control theweight average molecular weight of the hydrolyzed polymer product.

In various embodiments, removing the nitrogen protecting group from aprotected polylysine or polyornithine intermediate comprising repeatingunits of Formula 2 and hydrolyzing the polylysine or polyornithineintermediate are carried out concurrently. This can be by using the samereagent to remove the nitrogen protecting group and hydrolyze thepolylysine or polyornithine.

Definitions

As used herein, the “amino protecting groups” described herein aremoieties that block reaction at the protected amino group while beingeasily removed under conditions that are sufficiently mild so as not todisturb other substituents of the various compounds. For example, theamino protecting groups may be carbobenzyloxy (Cbz), t-butyloxycarbonyl(t-Boc), allyloxycarbonyl and the like. A variety of protecting groupsfor the amino group and the synthesis thereof may be found in“Protective Groups in Organic Synthesis” by T. W. Greene and P. G. M.Wuts, John Wiley & Sons, 1999.

The term “lysine-basis molecular weight” refers to the molecular weightof either the polylysine polymer itself, the deprotected polylysineintermediate, or the N^(ε)-protected polylysine intermediate asexpressed in terms of the equivalent weight of unprotected polylysinefree base. The term “ornithine-basis molecular weight” refers to themolecular weight of either the polyornithine polymer itself, thedeprotected polyornithine intermediate, or the N⁶⁷-protectedpolyornithine intermediate as expressed in terms of the equivalentweight of unprotected polyornithine free base. Expression of molecularweight on this basis is preferred because of the substantial differencesin molecular weight between repeating units of Formula 3 vs. Formula 2as well as differences between Formula 3 as depicted and the mineralacid salts thereof.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention.

The techniques used for the polymerization of the N-carboxyanhydrides(NCA) to polymers are known to those skilled in the art and are given indetail in the review article by M. Goodman and E. Peggion, Pure andApplied Chemistry, volume 53, p. 699, 1981 and the book by H. R.Kricheldorf “Alpha amino acids-N-Carboxyanhydrides and RelatedHeterocycles”, Springer Verlag (1987) and also the recent publicationsby Wendelmoed N. E. van Dijk-Wolthuis et al, Macromol. Chem. Phys.Volume 198, p. 3893-3906, 1997.

Example 1 Preparation of Poly-L-Lysine.HBr Polymer

Polymerization. N⁶⁸-Cbz-L-Lysine NCA (35 g, 0.114 mole) was dissolved in0.228 liter of 1,4-dioxane to make a 0.5 M solution. The NCA solutionwas transferred to a 1 liter three neck round bottom flask equipped withmechanical mixing and a water bath at a temperature of 25 to 30° C.Sodium methoxide (0.0114 moles, 11.4 mL of 1N solution) was placed in 25mL of 1,4-dioxane. The sodium methoxide solution was added to the NCAsolution in one portion with vigorous mixing. The polymerizationsolution was mixed for 2 hours and held at 25 to 30° C. for 18 to 24hours.

Deprotection of Cbz group. The protected polymer, Poly-Cbz-L-Lysine wasdiluted in 1,4-dioxane solution with 340 mL of 1,4-dioxane to make asolution having a 0.2 M concentration based on the amount of the NCAstarting material. To this solution was added 70 ml of a solution of 30%hydrobromic acid (HBr) in acetic acid (2 mL/g NCA starting material).This solution was added drop-wise with vigorous mixing in 20 minutes ata temperature of 25 to 30° C. The polymer began precipitating after 40mL of HBr in acetic acid was added. The resultant mixture was mixed at25 to 30° C. for 15 minutes to form a uniform slurry. Another 70 mL ofHBr in acetic acid (2 ml/g NCA) was added in one lot at 25 to 30° C. andmixed for 18 hours. The mixing was stopped and the precipitate wasallowed to settle for 15 minutes. The supernatant was decanted and 350mL of acetone was added to the precipitate and mixed for 5 minutes. Thesupernatant was then decanted. This washing step was repeated with 2×175mL of acetone. Acetone (200 mL) was added and the mixture was filteredon an 11 cm Buchner funnel, washed with 2×100 mL acetone and dried undera nitrogen atmosphere for 5 minutes. The resultant polymer was dissolvedin 350 mL deionized water and nitrogen was bubbled through the solutionto remove traces of benzyl bromide.

Dialysis/ultrafiltration and lyophilization (freeze drying). Thepoly-L-lysine.HBr solution was dialyzed or ultrafiltered against runningdeionized water using dialysis tubing having a molecular weight cut offof approximately 12K or a 5K membrane for ultrafiltration (UF) to removeoligomers and salts. The dialyzed or UF solution was collected, filteredthrough a 0.2 micron filter, and lyophilized (freeze dried) to get thesolid poly-L-lysine.HBr polymer. The yield was 73%. The ¹H NMR showedthe complete removal of the Cbz group (absence of aromatic peak). Theviscosity was measured in a 1 wt. % aqueous solution at 25° C. and theviscosity molecular weight was calculated as 26,750 Daltons.

Hydrolysis. The time required to hydrolyze the polymer using 48% aqueousHBr to the desired molecular weight (5,500-12,000 by GPC MALLS) wascalculated using the formula:

Time  required = [(Viscosity  Molecular  Weight − 21, 000) × 0.012] + 72(±10%)  HoursNumber  of  hours = [(Polymer  viscosity  Mol. Wt. − 21, 000) × 0.012] + 72  hours = (26, 750 − 21, 000) × 0.012 + 72  hours = 141  hours = 5.875  days

The polymer (16 g) was dissolved in 253 mL deionized water to form aclear solution, the solution was mixed for 10 minutes and 130 mL of 48%HBr (aqueous) was added and mixed for 141 hours.

The hydrolyzed poly-L-lysine.HBr solution was dialyzed or UF againstrunning deionized water using a dialysis tubing having a molecularweight cut off of approximately 12K or a 1K membrane for UF to removeoligomers and salts. The dialyzed or UF solution was collected, filteredthrough a 0.2 micron filter and lyophilized (freeze dried) to get thesolid poly-L-lysine.HBr polymer. The yield was 45.2%. The GPC MALLSmeasured molecular weight was 7,500 and the viscosity was measured in 1wt. % aqueous solution at 25° C. to calculate the viscosity molecularweight of 14,400 Daltons.

Example 2 Preparation of Poly-L-Lysine.HBr Polymer

Polymerization. N⁶⁸-Cbz-L-Lys NCA (350.0 g, 1.144 mole) was dissolved in2.29 liter of 1,4-dioxane to make a 0.5 M solution. The NCA solution wastransferred to a 12 liter three-neck round bottom flask equipped withmechanical mixing and a water bath at a temperature of 25 to 30° C.Sodium methoxide (114.4 ml of a 1 N solution, 0.1144 moles) was placedin 100 mL of 1,4-dioxane. The sodium methoxide solution was added to theNCA solution in one portion with vigorous mixing. The polymerizationsolution was mixed for 2 hours and held at 25 to 30° C. for 18 to 24hours.

Deprotection of Cbz group. The protected polymer, poly-Cbz-L-Lysine wasdiluted in 1,4-dioxane solution with 3,430 mL of 1,4-dioxane to make asolution having a 0.2 M concentration based on the amount of NCAstarting material. A solution of 30% HBr in acetic acid (700 mL, 2 mL/gNCA starting material) was added to the mixture drop-wise with vigorousmixing over 30 minutes at 25 to 30° C. The polymer started precipitatingafter about 350 mL of the HBr in acetic acid solution was added. Theresultant mixture was mixed at 25 to 30° C. for 15 minutes to form auniform slurry. Another 700 mL of 30% HBr in acetic acid solution (2ml/g NCA starting material) was added in one lot at 25-30° C. and mixedfor 18 hours. The mixing was stopped and the precipitate was allowed tosettle for 15 minutes. The supernatant was decanted, 3,500 mL of acetonewas added to the solids and mixed for 5 minutes, followed by decantingthe supernatant. This acetone addition, mixing, and decanting step wasrepeated twice with 2×1,750 mL of acetone. Acetone (2,000 mL) was addedand the polymer was filtered on an 18.5 cm Buchner funnel, washed with2×1,000 mL acetone, and allowed to dry under nitrogen atmosphere for 5minutes. The polymer was dissolved in 3,500 mL deionized water andnitrogen was bubbled through the solution to remove traces of benzylbromide.

Dialysis/ultrafiltration and lyophilization (freeze drying). Thepoly-L-lysine.HBr solution was dialyzed or UF against running deionizedwater using dialysis tubing having a molecular weight cut off ofapproximately 12K or a 5K membrane for UF to remove oligomers and salts.The dialyzed or UF solution was collected, filtered through a 0.2 micronfilter, and lyophilized (freeze dried) to get the solidpoly-L-lysine.HBr polymer. The yield was 75.5%. ¹H NMR showed thecomplete removal of the Cbz group (absence of aromatic peak). Theviscosity was measured in a 1 wt. % aqueous solution at 25° C. and theviscosity molecular weight was calculated as 27,700 Daltons.

Hydrolysis. The time required to hydrolyze the polymer using 48% aqueousHBr to the desired molecular weight (5,500-12,000 Daltons by GPC MALLS)was calculated as 152.4 hours (6.35 days) using the formula described inExample 1. The polymer (179.0 g) was dissolved in 2,828 mL deionizedwater to form a clear solution, mixed for 15 minutes, and 1,454 mL of48% aqueous HBr was added and mixed for 152.4 hours.

The hydrolyzed poly-L-lysine.HBr solution was dialyzed or UF againstrunning deionized water using dialysis tubing having a molecular weightcut off of approximately 12K or a 1K membrane for UF to remove oligomersand salts. The dialyzed or UF solution was collected, filtered through a0.2 micron filter and lyophilized (freeze dried) to get the solidpoly-L-lysine.HBr polymer. The yield was 53%. The molecular weightmeasured by GPC MALLS was 7,600 Daltons and the viscosity was measuredin a 1 wt. % aqueous solution at 25° C. to calculate the viscositymolecular weight of 14,200 Daltons.

Example 3 Preparation of Poly-L-Lysine.HBr Polymer

Polymerization. N⁶⁸-Cbz-L-Lysine NCA (7,500 g, 24.51 mole) wastransferred to a 100 gallon reactor equipped with a cooling/heatingjacket and dissolved in 49 liter of 1,4-dioxane to make an approximately0.5 M solution at a temperature of 25 to 30° C. A solution of 2,451 mLof 1 N sodium methoxide (2.451 moles) in methanol was prepared and addedto the NCA solution in one portion with vigorous mixing. Thepolymerization solution was mixed for 2 hours and then maintained at 25to 30° C. for 18 to 24 hours.

Deprotection of Cbz group. The protected polymer, poly-Cbz-L-Lysine in1,4-dioxane solution was diluted with 73.5 liter of 1,4-dioxane to makea solution having a 0.2M concentration based on the amount of NCAstarting material. A solution of 30% HBr in acetic acid (15,000 mL, 2mL/g NCA starting material) was added to the polymer solution slowlywith vigorous mixing over 60 minutes at a temperature of 25 to 30° C.The polymer began precipitating after the addition of about 7,500 mL ofHBr in acetic acid solution. The resultant mixture was mixed at atemperature of 25 to 30° C. for 45 minutes to form a uniform slurry.Another portion of HBr in acetic acid (15,000 mL, 2 mL/g NCA) was addedin one lot at a temperature of 25 to 30° C. and mixed for 18 hours. Themixing was stopped and the precipitate was allowed to settle for 30minutes. The supernatant was decanted and 75 liter of acetone was addedto the solids and mixed for 10 minutes, followed by decanting of thesupernatant. This step was repeated twice with 2×37.5 liter of acetone.Acetone (40 liter) was added and the polymer was filtered on aglass-lined contained filter. The polymer was washed with 2×20 literacetone and dried under nitrogen atmosphere for 15 minutes. The polymerwas dissolved in 75 liter of deionized water and nitrogen was bubbledthrough the solution to remove traces of benzyl bromide.

Dialysis/ultrafiltration and lyophilization (freeze drying). Thepoly-L-lysine.HBr solution was dialyzed or UF against running deionizedwater using dialysis tubing having a molecular weight cut off ofapproximately 12K or a 5K membrane for UF to remove oligomers and salts.The dialyzed or UF solution was collected, filtered through a 0.2 micronfilter, and lyophilized (freeze dried) to get the solidpoly-L-lysine.HBr polymer. The yield was 68.3%. ¹H NMR showed thecomplete removal of the Cbz group (absence of aromatic peak). Theviscosity was measured in a 1 wt. % aqueous solution at 25° C. and theviscosity molecular weight was calculated as 41,200.

Hydrolysis. The time required to hydrolyze the polymer using 48% aqueousHBr to the desired molecular weight (5,500-12,000 Daltons by GPC MALLS)was calculated as 13.1 days (314.4 hours) using the formula described inExample 1. The polymer (3,500 g) was dissolved in 55.3 liter deionizedwater to form a clear solution and mixed for 30 minutes. An aqueoussolution of 48% HBr (28.4 liter) was added and mixed for 314 hours.

The hydrolyzed poly-L-lysine.HBr solution was dialyzed or UF againstrunning deionized water using dialysis tubing having a molecular weightcut off of approximately 12K or a 1K membrane for UF to remove oligomersand salts. The dialyzed or UF solution was collected, filtered through a0.2 micron filter and lyophilized (freeze dried) to get the solidpoly-L-lysine.HBr polymer. The yield was 50.2%. The molecular weight wasmeasured by GPC MALLS as 9,300 Daltons and the viscosity was measured ina 1 wt. % aqueous solution at 25° C. and the viscosity molecular weightwas calculated as 17,300 Daltons.

Example 4 Preparation of Poly-L-Lysine.HBr Polymer

Polymerization. N⁶⁸-Cbz-L-Lysine NCA (25,490 g, 83.3 moles) wastransferred to a 200 gallon reactor equipped with cooling/heating jacketto maintain the temperature at 25 to 30° C. and dissolved in 155 literof 1,4-dioxane to make a solution having a NCA concentration of about0.5 M. A solution was prepared by combining 8,227 mL of 1N sodiummethoxide (8.227 moles) with methanol and this solution was added to theNCA solution in one portion with vigorous mixing. The polymerizationsolution was mixed for 2 hours and the temperature was maintained at 25to 30° C. for 18 to 24 hours.

Deprotection of Cbz group. The protected polymer, poly-Cbz-L-Lysine in a1,4-dioxane solution was diluted with 204 liter of 1,4-dioxane to make asolution having a concentration of about 0.2M based on the amount of NCAstarting material. A solution of 30% HBr in acetic acid (50.4 liter, 2ml/g NCA starting material) was added into the polymer solution slowlywith vigorous mixing in 60 minutes at a temperature of 25 to 30° C. Thepolymer started precipitating after about 25 liters of the HBr in aceticacid solution was added. The resultant mixture was mixed at atemperature of 25 to 30° C. for 30 minutes to form a uniform slurry. Asolution of HBr in acetic acid (50.4 liter, 2 ml/g NCA startingmaterial) was added in one lot at a temperature of 25 to 30° C. andmixed for 18 hours. The mixing was stopped and the precipitate wasallowed to settle for 30 minutes. Ice-cold acetone (383 liter) was addedto the solids, mixed for about 30 minutes, and filtered through aglass-lined filter. The solids were washed with 3×172 liter of acetoneand allowed to dry under a nitrogen atmosphere for 15 minutes. Thepolymer was dissolved in about 235 liter deionized water and nitrogenwas bubbled through the solution to remove traces of benzyl bromide.

Dialysis/ultrafiltration and lyophilization (freeze drying). Thepoly-L-lysine.HBr solution was dialyzed or UF against running deionizedwater using dialysis tubing having a molecular weight cut off ofapproximately 12K or a 5K membrane for UF to remove oligomers and salts.The dialyzed or UF solution was collected, filtered through a 0.2 micronfilter, and 150 mL was lyophilized (due to a very large volume of thesolution to be lyophilized) to get the solid weight forpoly-L-lysine.HBr polymer. The total solids weight was calculated basedon the estimated total volume. The yield was estimated to be about 55%.¹H NMR showed the complete removal of the Cbz group (absence of aromaticpeak). The viscosity of the polymer in a 1 wt. % aqueous solution wasmeasured at 25° C. and the viscosity molecular weight was calculated as32,360 Daltons.

Hydrolysis. The time required to hydrolyze the polymer using 48% aqueousHBr to the desired molecular weight (5,500-12,000 Daltons by GPC MALLS)was calculated as 208.3 hours (9.17 days) using the formula described inExample 1. An aqueous 48% HBr was added to the polymer solution in anamount to produce a 0.3 M HBr concentration. This solution was mixed for220 hours.

The hydrolyzed poly-L-lysine.HBr solution was dialyzed or UF againstrunning deionized water using a dialysis tubing having a molecularweight cut off of approximately 12K or a 1K membrane for UF to removeoligomers and salts. The dialyzed or UF solution was collected, filteredthrough a 0.2 micron filter, and lyophilized (freeze dried) to get thesolid poly-L-lysine.HBr polymer. The yield was 52%. The molecular weightmeasured by GPC MALLS was 8,100 Daltons and the viscosity was measuredin a 1 wt. % aqueous solution at 25° C. and the viscosity molecularweight was calculated as 14,800 Daltons.

When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above processes withoutdeparting from the scope of the invention, it is intended that allmatter contained in the above description shall be interpreted asillustrative and not in a limiting sense.

1. A process for the preparation of a polylysine polymer comprisingrepeating units corresponding to Formula 3 or a salt thereof and havinga lysine-basis weight average molecular weight from about 5,500 to about12,000 Daltons, the process comprising: hydrolyzing a polylysineintermediate comprising repeating units corresponding to Formula 3 or asalt thereof and having a lysine-basis weight average molecular weightfrom about 12,500 to about 22,000 Daltons, wherein Formula 3 has thestructure:

n is 4; and wherein said polylysine polymer is a homopolymer of lysine.2. The process as set forth in claim 1 wherein preparation of saidpolylysine intermediate comprises removing a nitrogen protecting groupfrom a protected polylysine intermediate comprising repeating units ofFormula 2 or a salt thereof and having a lysine-basis weight averagemolecular weight from about 12,500 Daltons to about 22,000 Daltons,wherein Formula 2 has the structure:

R₁ is an amino protecting group; and n is
 4. 3. The process as set forthin claim 2 wherein preparation of said protected polylysine intermediatecomprises polymerizing an N-carboxyanhydride compound of Formula 1,wherein Formula 1 has the structure:

R₁ is an amino protecting group; and n is
 4. 4. The process of claim 2wherein hydrolyzing the polylysine intermediate and removing thenitrogen protecting group from the protected polylysine intermediate arecarried out concurrently.
 5. The process of claim 2 wherein the aminoprotecting group is carbobenzyloxy, t-butoxycarbonyl, orallyloxycarbonyl.
 6. The process of claim 3 wherein the polymerizationis conducted in the presence of an initiator.
 7. The process of claim 6wherein the initiator comprises a metal alkoxide.
 8. The process ofclaim 3 wherein the polymerization is conducted in an organic solventmedium and deprotection of the N-protected polylysine intermediatecomprises introducing a combination of a mineral acid and an organicacid into said polymerization medium, thereby forming a precipitatecomprising repeating units consisting of the N^(ε)-salt of the repeatingunits of Formula 3 and said mineral acid.
 9. The process of claim 3comprising removing oligomers and other low molecular weight componentsfrom a solution comprising the deprotected polylysine intermediate priorto hydrolysis of said deprotected intermediate.
 10. The process of claim9 wherein the deprotected polylysine intermediate and associatedoligomers and/or other low molecular weight components are taken up intoan aqueous medium, and the aqueous medium containing the deprotectedintermediate polymer and oligomers and/or other low molecular weightcomponents is contacted with a surface of a dialysis membrane having anaqueous medium on the opposite surface thereof into which oligomers andother low molecular weight components are received in the dialysis,thereby producing a retentate comprising deprotected polylysineintermediate of reduced oligomer content.
 11. The process of claim 10wherein the retentate is lyophilized to yield solid deprotectedpolylysine.
 12. The process of claim 11 wherein the solid deprotectedpolylysine is taken up into an aqueous solution wherein it is contactedwith a mineral acid for the hydrolysis.
 13. The process of claim 2wherein the hydrolysis of the deprotected polylysine intermediate iscarried out for a reaction time calculated from the viscosity molecularweight of the deprotected polylysine intermediate.
 14. The process ofclaim 3 wherein the polymerization of the N-carboxyanhydride compound isconducted in a polymerization reaction medium comprising a nonpolarsolvent.
 15. The process of claim 3 wherein the yield of polylysinepolymer is greater than about 45% based on the quantity of theN-carboxyanhydride starting material.
 16. The process of claim 1 whereinthe polylysine polymer is poly-L-lysine, poly-D-lysine, or a racemicmixture thereof.
 17. The process of claim 13 wherein an algorithm isused to calculate the hydrolysis reaction time, the algorithm being:Time Required =[(viscosity molecular weight of unhydrolyzedpolymer−21,000) ×0.012]+72(±10%) hours.
 18. The process of claim 1wherein the polylysine polymer comprising repeating units correspondingto Formula 3 or a salt thereof and having a lysine-basis weight averagemolecular weight from about 5,500 to about 12,000 Daltons has apolydispersity index of about 1.2 to about 1.5.
 19. A process for thepreparation of a polylysine or polyornithine polymer comprisingrepeating units corresponding to Formula 3 or a salt thereof and havinga lysine-basis or ornithine-basis weight average molecular weight fromabout 5,500 to about 12,000 Daltons, the process comprising: hydrolyzinga polylysine or polyornithine intermediate comprising repeating unitscorresponding to Formula 3 or a salt thereof and having a lysine-basisor ornithine-basis weight average molecular weight from about 12,500 toabout 22,000 Daltons, wherein Formula 3 has the structure:

n is 3 or 4; and the hydrolysis of the polylysine or polyornithineintermediate is carried out for a reaction time calculated according toan algorithm, the algorithm being:Time Required =[(viscosity molecular weight of unhydrolyzedpolymer−21,000) ×0.012]+72(±10%) hours.